Cells expressing modified T cell receptor

ABSTRACT

This invention provides a cell presenting at least one T cell receptor (TCR) anchored to the membrane by a transmembrane sequence, said TCR comprising an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a division of U.S. application Ser. No. 11/597,252filed Apr. 28, 2009, now U.S. Pat. No. 8,361,794, which is a NationalStage application of co-pending PCT application PCT/GB2005/002570 filedJun. 29, 2005, which was published in English under PCT Article 21(2) onJan. 5, 2006, and which claims the benefit of Great Britain patentapplications Serial No. GB 0414499.4 filed Jun. 29, 2004; Serial No.0421831.9 filed Oct. 1, 2004; and Serial No. 0511123.2 filed Jun. 1,2005. These applications are incorporated herein by reference in theirentireties.

REFERENCE TO A SEQUENCE LISTING

This application incorporates by reference the contents of a 80.6 kbtext file created on Oct. 29, 2009 and named“SN11597252_sequencelisting.txt, which is the sequence listing for thisapplication.

FIELD OF THE INVENTION

The present invention relates to cells, particularly T cells, expressingmodified T cell receptors (TCRs), their preparation, and their use intherapy.

BACKGROUND TO THE INVENTION

Native TCRs

As is described in, for example, WO 99/60120 TCRs mediate therecognition of specific Major Histocompatibility Complex (MHC)-peptidecomplexes by T cells and, as such, are essential to the functioning ofthe cellular arm of the immune system.

Antibodies and TCRs are the only two types of molecules which recogniseantigens in a specific manner, and thus the TCR is the only receptor forparticular peptide antigens presented in MHC, the alien peptide oftenbeing the only sign of an abnormality within a cell. T cell recognitionoccurs when a T-cell and an antigen presenting cell (APC) are in directphysical contact, and is initiated by ligation of antigen-specific TCRswith pMHC complexes.

The native TCR is a heterodimeric cell surface protein of theimmunoglobulin superfamily which is associated with invariant proteinsof the CD3 complex involved in mediating signal transduction. TCRs existin αβ and γδ forms, which are structurally similar but have quitedistinct anatomical locations and probably functions. The MHC class Iand class II ligands are also immunoglobulin superfamily proteins butare specialised for antigen presentation, with a highly polymorphicpeptide binding site which enables them to present a diverse array ofshort peptide fragments at the APC cell surface.

Two further classes of proteins are known to be capable of functioningas TCR ligands. (1) CD1 antigens are MHC class I-related molecules whosegenes are located on a different chromosome from the classical MHC classI and class II antigens. CD1 molecules are capable of presenting peptideand non-peptide (eg lipid, glycolipid) moieties to T cells in a manneranalogous to conventional class I and class II-MHC-pep complexes. See,for example (Barclay et al, (1997) The Leucocyte Antigen Factsbook2^(nd) Edition, Academic Press) and (Bauer (1997) Eur J Immunol 27 (6)1366-1373)) (2) Bacterial superantigens are soluble toxins which arecapable of binding both class II MHC molecules and a subset of TCRs.(Fraser (1989) Nature 339 221-223) Many superantigens exhibitspecificity for one or two Vbeta segments, whereas others exhibit morepromiscuous binding. In any event, superantigens are capable ofeliciting an enhanced immune response by virtue of their ability tostimulate subsets of T cells in a polyclonal fashion.

The extracellular portion of native heterodimeric αβ and γδ TCRs consistof two polypeptides each of which has a membrane-proximal extracellularconstant domain, and a membrane-distal variable region. Each of theextracellular constant domain and variable region includes anintra-chain disulfide bond. The variable regions contain the highlypolymorphic loops analogous to the complementarity determining regions(CDRs) of antibodies. CDR3 of αβ TCRs interact with the peptidepresented by MHC, and CDRs 1 and 2 of αβ TCRs interact with the peptideand the MHC. The diversity of TCR sequences is generated via somaticrearrangement of linked variable (V), diversity (D), joining (J), andconstant genes, the genes products thereof making up the variableregion.

Functional α and γ chain polypeptides are formed by rearranged V-J-Cdomains, whereas β and δ chains consist of V-D-J-C domains. (See FIG. 1)Each functional TCR possessing one of the possible variants of eachdomain. (See FIGS. 7 and 8 for the DNA sequences of all known TCR C andV domains from TCR α and β chains respectively, also see (LeFranc et al,(2001) The T cell receptor Factsbook, Academic Press) for a completelisting of the DNA and amino acid sequences of all known TCR domains) Afurther level of diversity is introduced to αβ TCRs by the in-vivorecombination of shortened TCR domains. The extracellular constantdomain has a membrane proximal motif and an immunoglobulin motif. Thereare single α and δ chain constant domains, known as TRAC and TRDCrespectively. The β chain constant domain is composed of one of twodifferent β constant domains, known as TRBC1 and TRBC2 (IMGTnomenclature). There are four amino acid changes between these βconstant domains, three of which are in exon 1 of TRBC1 and TRBC2:N₄K₅->K₄N₅ and F₃₇->Y, the final amino acid change between the two TCR βchain constant regions being in exon 3 of TRBC1 and TRBC2: V₁->E. (IMGTnumbering, differences TRBC1->TRBC2) The constant γ domain is composedof one of either TRGC1, TRGC2(2×) or TRGC2(3×). The two TRGC2 constantdomains differ only in the number of copies of the amino acids encodedby exon 2 of this gene that are present. TCR constant domains include atransmembrane sequence, the amino acids of which anchor the TCR chainsinto the cell surface membrane. There are 46 different TRAY domains and56 TRBV domains. 52 different functional genes encode the TRAJ domains,whereas 12-13 functional genes encode the TRBJ domains. 2 differentfunctional genes encode the TRBD domains.

The extent of each of the TCR extracellular constant domains, bounded bythe transmembrane sequences, is somewhat variable. However, a personskilled in the art can readily determine the position of the domainboundaries using a reference such as The T Cell Receptor Facts Book,Lefranc & Lefranc, Publ. Academic Press 2001.

Immunotherapy

Immunotherapy involves enhancing the immune response of a patient tocancerous or infected cells. Active immunotherapy is carried out bystimulation of the endogenous immune system of tumour bearing patients.Passive, or adoptive, immunotherapy involves the transfer of immunecompetent cells into the patient. (Paul (2002) Curr Gene Therapy 291-100) There are three broad approaches to adoptive immunotherapy whichhave been applied in the clinic for the treatment of metastaticdiseases; lymphokine-activated killer (LAK) cells, auto-lymphocytotherapy (ALT) and tumour-infiltrating lymphocutes (TIL). (Paul (2002)Curr Gene Therapy 2 91-100).

A recent proposed variation of T cell adoptive therapy is the use ofgene therapy techniques to introduce TCRs specific for knowncancer-specific MHC-peptide complexes into the T cells of cancerpatients. For example, (WO 01/55366) discloses retrovirus-based methodsfor transfecting, preferably, T cells with heterologous TCRs. Thisdocument states that these transfected cells could be used for eitherthe cell surface display of TCR variants as a means of identifying highaffinity TCRs or for immunotherapy. Methods for the molecular cloning ofcDNA of a human p53-specific, HLA restricted murine TCR and the transferof this cDNA to human T cells are described in published US patentapplication no. 20020064521. This document states that the expression ofthis murine TCR results in the recognition of endogenously processedhuman p53 expressed in tumour cells pulsed with the p53-derived peptide149-157 presented by HLA A*0201 and claims the use of the murine TCR inanti-cancer adoptive immunotherapy. However, the concentration ofpeptide pulsing required achieving half maximal T cell stimulation ofthe transfected T cells was approximately 250 times that required by Tcells expressing solely the murine TCR. As the authors noted “Thedifference in level of peptide sensitivity is what might be expected ofa transfectant line that contained multiple different TCR heterodimersas a result of independent association of all four expressed hu and muTCR chains.”

There are also a number of recent papers relating to T cell adoptivetherapy. In one study (Rosenberg (1988) N Engl J Med 319 (25) 1676-80)lymphocytes from melanomas were expanded in vitro and thesetumor-infiltrating lymphocytes, in combination with IL-2 were used totreat 20 patients with metastatic melanoma by means of adoptivetransfer. The authors note that objective regression of the cancer wasobserved in 9 of 15 patients (60 percent) who had not previously beentreated with interleukin-2 and in 2 of 5 patients (40 percent) in whomprevious therapy with interleukin-2 had failed. Regression of canceroccurred in the lungs, liver, bone, skin, and subcutaneous sites andlasted from 2 to more than 13 months. A further study describes theadministration of an expanded population of Melan-A specific cytotoxic Tcells to eight patients with refractory malignant melanoma. These Tcells were administered by i.v. infusion at fortnightly intervals,accompanied by s.c. administration of IL-2. The T cell infusions werewell tolerated with clinical responses noted as one partial, one mixedwith shrinkage of one metastatic deposit and one no change (12 months)among the eight patients. (Meidenbauer (2003) J Immunol 170 2161-2169)As noted in this study, recent advances regarding the in vitrostimulation T cells for the generation of cell populations suitable forT cell adoptive therapy have made this approach more practical. See, forexample (Oelke (2000) Clin Cancer Res 6 1997-2005) and (Szmania (2001)Blood 98 505-12).

SUMMARY OF THE INVENTION

The present invention relates to cells presenting at least one T cellreceptor (TCR) anchored to the membrane by a transmembrane sequence,said TCR comprising an interchain disulfide bond between extracellularconstant domain residues which is not present in native TCRs. Such Tcells are expected to be particularly suited for use in T cell adoptiveimmunotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 illustrates the domains that comprise TCR α and β chains: wherein“S” denotes the signal peptide, “V” denotes the variable domain, “J”denotes the joining domain, D denotes the diversity domain, and “C”denotes the constant domain which contains the transmembrane sequence;

FIG. 2 illustrates the structure of a cell surface TCR containing anon-native interchain disulfide bond;

FIGS. 3 a and 3 b show respectively the nucleic acid sequences of the a(SEQ ID NO:34) and β (SEQ ID NO:35) chains of a soluble A6 TCR, mutatedso as to introduce a cysteine codon. The shading indicates theintroduced cysteine codons;

FIG. 4 a shows the amino acid sequence (SEQ ID NO:36) encoded by the DNAsequence of FIG. 3 a, including the T₄₈→C mutation (underlined) used toproduce the novel disulfide inter-chain bond, and FIG. 4 b shows theamino acid sequence (SEQ ID NO:37) encoded by the DNA sequence of FIG. 3b, including the S₅₇→C mutation (underlined) used to produce the noveldisulfide inter-chain bond;

FIG. 5 graphically illustrates the PCR reactions required to produce aDNA sequence encoding a full-length disulfide-linked A6 Tax TCR usingDNA encoding soluble disulfide-linked A6 Tax TCR and wild-type A6 TaxTCR as templates;

FIG. 6 a shows the nucleic acid (SEQ ID NO:38) and protein sequences(SEQ ID NO:39) of the membrane anchored α chain of A6 TCR, mutated so asto introduce a new cysteine codon and mutate the Cys involved in formingthe native inter-chain disulfide bridge to Ser. The first shadingindicates the introduced cysteine codon; the underlined Ser codonindicates the position of the Cys->Ser mutation disrupting the capacityto form the native inter-chain disulfide link.

FIG. 6 b shows nucleic acid (SEQ ID NO:40) and protein sequences (SEQ IDNO:41) of the membrane anchored β chain of A6 TCR, using the nativeconstant domain, TRBC2 (nomenclature according to the IMGT format asdescribed in (LeFranc et al, (2001) The T cell receptor Factsbook,Academic Press), mutated so as to introduce a new cysteine codon andmutate the Cys involved in forming the native inter-chain disulfidebridge to Ser. The first shading indicates the introduced cysteinecodon; the underlined Ser codon indicates the position of the Cys->Sermutation disrupting the capacity to form the native inter-chaindi-sulfide link.

FIGS. 7 a-7 h detail the DNA sequence of all known TCR α chain constantand variable domains (SEQ ID NOS: 41-89.

FIGS. 8 a-8 j detail the DNA sequence of all known TCR β chain constantand variable domains SEQ ID NOS:90-145.

FIGS. 9 a and 9 b show respectively the DNA sequences of the α (SEQ IDNO:146) and β (SEQ ID NO:147) chains of a soluble AH-1.23 TCR, mutatedso as to introduce a novel cysteine codon (indicated by shading).

FIGS. 10 a and 10 b show respectively the AH-1.23 TCR α (SEQ ID NO:148)and P (SEQ ID NO:149) chain extracellular amino acid sequences producedfrom the DNA sequences of FIGS. 9 a and 9 b.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a cell presenting at least one T cell receptor(TCR) anchored to the membrane by a transmembrane sequence, said TCRcomprising an interchain disulfide bond between extracellular constantdomain residues which is not present in native TCRs.

As noted above native TCRs exist in αβ and γδ forms, the presentinvention encompasses cells presenting either of these TCR forms,wherein said TCR comprises an interchain disulfide bond betweenextracellular constant domain residues which is not present in nativeTCRs.

Another embodiment provides a cell presenting at least one αβ T cellreceptor (TCR) anchored to the membrane by a transmembrane sequence,said TCR comprising a disulfide bond between α and β extracellularconstant domain residues which is not present in native TCRs.

The presence of the novel cysteine residues (creating the noveldisulfide bond) in the transfected heterodimeric TCR (dTCR) chainsfavour the production of the desired transfected TCRs over TCRscomprising a native TCR chain associated with a transfected TCR chain.Without wishing to be bound by theory, this result is interpreted asbeing due to the two transfected TCR chains preferentiallyself-associating due to the formation of the novel inter-chain disulfidebond between the introduced cysteine residues. The formation of any TCRcomprising a mismatched pair of TCR chains (one native and one from thetransfected TCR) may be further inhibited by ensuring the transfectedchains lack the cysteine residue involved in the formation of the nativeinter-chain disulfide bond. Cells expressed such transfected TCR chainstherefore provide a preferred embodiment of the invention.

Use of a single chain TCR (scTCR) in accordance with the invention alsoavoids formation of mismatched pairs.

The αβ TCRs which comprise a disulfide bond between α and βextracellular constant domain residues which is not present in nativeTCRs presented by the cells of the invention are targeting moieties. TheTCRs of the invention target TCR ligands such as peptide-MHC orCD1-antigen complexes. As such, it would be desirable if the affinity ofthese TCR could be altered. For example it may be desirable if these TCRhad a higher affinity and/or a slower off-rate for the TCR ligands thannative TCRs specific for that ligand. The inventors co-pendingapplication WO 2004/044004 details methods of producing and testing TCRshaving a higher affinity and/or a slower off-rate for the TCR ligandthan native TCRs specific for that ligand.

The TCR functionality of cells transfected to express and present themembrane anchored scTCRs and dTCRs may be tested by confirming thattransfected cells bind to the relevant TCR ligand (pMHC complex,CD1-antigen complex, superantigen or superantigen/pMHC complex)—if itbinds, then the requirement is met. The binding of the transfected cellsto a TCR ligand can be detected by a number of methods. These includeattaching a detectable label to the TCR ligand. For example, where themethod uses pMHC tetramers, the pMHC may include a fluorescent label.Example 6 herein provides a detailed description of the methods requiredto analyse the binding of cells transfected to express disulfide-linkedTCRs to MHC-peptide complexes. This method is equally applicable to thestudy of TCR/CD1 interactions. In order to apply this method to thestudy of TCR/CD1 interactions soluble forms of CD1 are required, theproduction of which are described in (Bauer (1997) Eur J Immunol 27 (6)1366-1373).

The Cell Membrane Anchored dTCR

In the case of a dTCR, the TCR □ and □ chains may each comprise atransmembrane sequence, fused at its N terminus to an extracellularconstant domain sequence, in turn fused at its N terminus to a variableregion sequence. Furthermore, at least the said sequences of the TCR αand β chains other than the complementarity determining regions of thevariable region, may correspond to human TCR α and β sequences.

The Cell Membrane Anchored scTCR

In the case of an scTCR, the scTCR comprises

-   -   (i) a first segment constituted by an α chain variable region        sequence fused to the N terminus of an α chain extracellular        constant domain sequence, and a second segment constituted by a        β chain variable region sequence fused to the N terminus of a        sequence β chain extracellular constant and transmembrane        sequence, and a linker sequence linking the C terminus of the        first segment to the N terminus of the second segment, or    -   (ii) a first segment constituted by a TCR β chain variable        region sequence fused to the N terminus of a β chain        extracellular constant domain sequence, and a second segment        constituted by an α chain variable region sequence fused to the        N terminus of a sequence α chain extracellular constant and        transmembrane sequence, and a linker sequence linking the C        terminus of the first segment to the N terminus of the second        segment.

Again, in the scTCR embodiment, the said sequences of the TCR α and βchains other than the complementarity determining regions of thevariable region, correspond to human TCR α and β sequences.

Linker in the Membrane Anchored scTCR Polypeptide

For the cell presented scTCRs of the present invention, a linkersequence links the first and second TCR segments, to form a singlepolypeptide strand. The linker sequence may, for example, have theformula -P-AA-P- wherein P is proline and AA represents an amino acidsequence wherein the amino acids are glycine and serine.

For the cell presented scTCRs of the present invention to bind to a TCRligand, such as MHC-peptide or CD1-antigen complexes, the first andsecond segments must be paired so that the variable region sequencesthereof are orientated for such binding. Hence the linker should havesufficient length to span the distance between the C terminus of thefirst segment and the N terminus of the second segment, or vice versa.On the other hand excessive linker length should preferably be avoided,in case the end of the linker at the N-terminal variable region sequenceblocks or reduces bonding of the scTCR to the target ligand.

For example, in the case where the constant region extracellularsequences present in the first segment correspond to the constantregions of the α and β chains of a native TCR truncated at their Ctermini such that the cysteine residue that forms the native interchaindisulfide bond of the TCR is excluded, and the linker sequence links theC terminus of the first segment to the N terminus of the second segment,the linker may consist of from 26 to 41, for example 29, 30, 31 or 32amino acids, and a particular linker has the formula -PGGG-(SGGGG)₅-P-wherein P is proline, G is glycine and S is serine (SEQ ID NO:1).

The Cell Membrane Anchored scTCR and dTCR

As mentioned above, preferred embodiments the dTCR or scTCR α and βchain sequences correspond to human TCR α and β sequences, with theexception of the complementarity determining regions (CDRs) of thevariable regions which may or may not correspond to human CDR sequences.However, correspondence between such sequences need not be 1:1 on anamino acid level. N- or C-truncation, and/or amino acid deletion and/orsubstitution relative to corresponding human TCR sequences isacceptable, provided the overall result is a cell membrane anchored TCRcomprising mutual orientation of the α and β variable region sequencesis as in native αβ T cell receptors respectively. In particular, becausethe constant domain extracellular sequences are not directly involved incontacts with the ligand to which the cell membrane anchored scTCR ordTCR binds, they may be shorter than, or may contain substitutions ordeletions relative to, extracellular constant domain sequences of nativeTCRs.

Included in the scope of this invention are cells presenting membraneanchored TCRs comprising amino acids encoded by any appropriatecombination of the nucleic acid sequences corresponding to thosedisclosed in FIGS. 7 and 8. As is known to those skilled in the art,TCRs can also be produced by combination of amino acid sequences encodedby truncated variants of the sequences disclosed in FIGS. 7 & 8. SuchTCRs form an additional embodiment of the present invention. Alsoincluded within the scope of this invention are membrane anchored TCRsencoded by any variants of these nucleic acid molecules.

Usually, cells according to the invention will present a plurality ofthe said scTCR or dTCR (the exogenous TCRs). Each of the plurality ofthe said scTCRs or dTCRs is preferably identical, but if the cell is aT-cell, it may also present some native (endogenous) TCRs, residuallyencoded by the T cell chromosomes.

Another preferred embodiment provides T cells having the said membraneanchored scTCR or dTCR, or a plurality thereof. In a further preferredembodiment these T cells are cytotoxic T cells.

Another preferred embodiment provides cells that reduces the cellular orpro-inflammatory arms of an auto-immune response having the saidmembrane anchored scTCR or dTCR, or a plurality thereof. Examples ofsuch cells, include, but are not limited to macrophages, γδ T cells, Th3T cells, Tr1 T cells, NK T cells, macrophages and regulatory T cells. Ina further preferred embodiment these cells are regulatory T cells.

Regulatory T cells are characterised by the cell-surface expression ofCD4 and CD25. (Bluestone and Tang Proc Natl Acad Sci USA. 2004 101 Suppl2: 14622-6.) provides a review of regulatory T cells.

In a further embodiment of the invention the cells present scTCR or dTCRwhich contains a covalent disulfide bond linking a residue of theimmunoglobulin region of the constant domain of the α chain to a residueof the immunoglobulin region of the constant domain of the β chain.

A further embodiment of the invention provides a cell presenting scTCRor dTCR wherein in the said TCR an interchain disulfide bond in nativeTCR is not present.

A further embodiment of the invention provides a cell presenting scTCRor dTCR wherein in the said TCR cysteine residues which form the nativeinterchain disulfide bond are substituted to another residue.

A further embodiment of the invention provides a cell presenting scTCRor dTCR wherein in the said TCR cysteine residues which form the nativeinterchain disulfide bond are substituted to serine or alanine.

A further embodiment of the invention provides a cell presenting scTCRor dTCR wherein in the said TCR an unpaired cysteine residue present innative TCR β chain is not present.

Inter-Chain Disulfide Bond

A principal characterising feature of the cell membrane anchored scTCRsand dTCRs of the present invention, is a disulfide bond between theconstant region extracellular sequences of the dTCR polypeptide pair orfirst and second segments of the scTCR polypeptide. That bond maycorrespond to the native inter-chain disulfide bond present in nativedimeric αβ TCRs, or may have no counterpart in native TCRs, beingbetween cysteines specifically incorporated into the constant regionextracellular sequences of dTCR polypeptide pair or first and secondsegments of the scTCR polypeptide. In some cases, both a native and anon-native disulfide bond may be desirable.

The position of the disulfide bond is subject to the requirement thatthe variable region sequences of dTCR polypeptide pair or first andsecond segments of the scTCR polypeptide are mutually orientatedsubstantially as in native αβ T cell receptors.

The disulfide bond may be formed by mutating non-cysteine residues onthe first and second segments to cysteine, and causing the bond to beformed between the mutated residues. Residues whose respective β carbonsare approximately 6 Å (0.6 nm) or less, and preferably in the range 3.5Å (0.35 nm) to 5.9 Å (0.59 nm) apart in the native TCR are preferred,such that a disulfide bond can be formed between cysteine residuesintroduced in place of the native residues. It is preferred if thedisulfide bond is between residues in the constant immunoglobulinregion, although it could be between residues of the membrane proximalregion. Preferred sites where cysteines can be introduced to form thedisulfide bond are the following residues in exon 1 of TRAC*01 for theTCR α chain and TRBC1*01 or TRBC2*01 for the TCR β chain:

TCR α chain TCR β chain Native β carbon separation (nm) Thr 48 Ser 570.473 Thr 45 Ser 77 0.533 Tyr 10 Ser 17 0.359 Thr 45 Asp 59 0.560 Ser 15Glu 15 0.59

Now that the residues in human TCRs which can be mutated into cysteineresidues to form a new interchain disulfide bond in cell membrane bounddTCRs or scTCRs according to the invention have been identified, thoseof skill in the art will be able to mutate TCRs of other species in thesame way to produce a dTCR or scTCR of that species for cell membranebound expression. In humans, the skilled person merely needs to look forthe following motifs in the respective TCR chains to identify theresidue to be mutated (the shaded residue is the residue for mutation toa cysteine).

αChain Thr 48:

αChain Thr 45:

αChain Tyr 10:

αChain Ser 15:

β Chain Ser 57:

β Chain Ser 77:

β Chain Ser 17:

β Chain Asp 59:

β Chain Glu 15:

In other species, the TCR chains may not have a region which has 100%identity to the above motifs. However, those of skill in the art will beable to use the above motifs to identify the equivalent part of the TCRα or β chain and hence the residue to be mutated to cysteine. Alignmenttechniques may be used in this respect. For example, ClustalW, availableon the European Bioinformatics Institute website atwww.ebi.ac.uk/index.html can be used to compare the motifs above to aparticular TCR chain sequence in order to locate the relevant part ofthe TCR sequence for mutation.

The present invention includes within its scope cell membrane bound αβscTCRs and dTCRs, as well as those of other mammals, including, but notlimited to, mouse, rat, pig, goat and sheep. As mentioned above, thoseof skill in the art will be able to determine sites equivalent to theabove-described human sites at which cysteine residues can be introducedto form an inter-chain disulfide bond. For example, the following showsthe amino acid sequences of the mouse Cα and Cβ soluble domains,together with motifs showing the murine residues equivalent to the humanresidues mentioned above that can be mutated to cysteines to form a TCRinterchain disulfide bond (where the relevant residues are shaded):

Mouse Cα soluble domain: (SEQ ID NO: 11)PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVP Mouse Cβ soluble domain:(SEQ ID NO: 12) EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGREVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRAD Murine equivalent of human α Chain Thr 48:(SEQ ID NO: 13)

Murine equivalent of human α Chain Thr 45: (SEQ ID NO: 14)

Murine equivalent of human α Chain Tyr 10: (SEQ ID NO: 15)

Murine equivalent of human α Chain Ser 15: (SEQ ID NO: 16)

Murine equivalent of human β Chain Ser 57: (SEQ ID NO: 17)

Murine equivalent of human β Chain Ser 77: (SEQ ID NO: 18)

Murine equivalent of human β Chain Ser 17: (SEQ ID NO: 19)

Murine equivalent of human β Chain Asp 59: (SEQ ID NO: 20)

Murine equivalent of human β Chain Glu 15: (SEQ ID NO: 21)

A nucleic acid molecule or molecules comprising a sequence or sequencesencoding a membrane anchored scTCR or dTCR are also provided, as arevectors comprising said nucleic acid molecules. Included in the scope ofthis invention are nucleic acid sequences encoding membrane anchored TCRcomprising any appropriate combination of nucleic acid sequencecorresponding to those disclosed in FIGS. 7 and 8. As is known to thoseskilled in the art, TCRs can also be produced that comprise combinationsof amino acids encoded by truncated variants of the nucleic sequencesdisclosed in FIGS. 7 and 8, such nucleic acid sequences form anadditional embodiment of the present invention. Also included within thescope of this invention are variants of these nucleic acid moleculesthat encode membrane anchored TCRs.

The nucleic acid or nucleic acids encoding TCRs of the invention may beprovided in a form which has been adapted for expression in a prokaryoteor eukaryote host cell. Suitable host cells include, but are not limitedto, bacterial, yeast, mammalian or insect cells. For example, the hostcell may be a human T cell or a human haematopoietic stem cell.

Such adapted nucleic acid or nucleic acids is/are mutated to reflect thecodon preference of the host cell in to which it is introduced. Themutations introduced are silent mutations which do not affect the aminoacid sequence of the polypeptide or polypeptides thereby encoded.GeneArt (Regensburg, Germany) offer a suitable nucleic acid optimisationservice (GeneOptimizer™). WO 2004/059556, owned by GeneArt, providesfurther details of the optimisation process. Nucleic acid complementaryto any such adapted nucleic acid sequence or a RNA sequencecorresponding thereto also forms part of this invention. Furthermore, aswill be obvious to those skilled in the art such nucleic acid or nucleicacids encoding TCRs of the invention may also comprise non-coding(intron) sequences.

As will be obvious to those skilled in the art such full-length TCRchain DNA sequences encode for the following sequences:

-   -   A leader sequence and the extracellular, transmembrane, and        cytoplasmic TCR sequences.

A method for obtaining a cell expressing a membrane anchored scTCR ordTCR is also provided, said method comprises incubating a host cellharbouring a vector encoding the membrane anchored scTCR or dTCR underconditions causing expression of the scTCR or dTCR.

Preparation of Cells Expressing TCRs Comprising a Non-Native DisulfideInterchain Bond

Another embodiment provides a method for the preparation of cells of theinvention said method comprising:

-   -   (a) isolation of a population of cells, preferably a population        of T cells    -   (b) in vitro transfection of said population of cells with an        expression vector encoding a TCR of the invention specific for a        target cell,    -   (c) optional in vitro growth of the transfected cells.

In a preferred embodiment the population of cells is isolated from apatient to be treated by a method of directing said cells to apopulation of target cells.

The following provides details of the isolation, transformation andoptional in-vitro growth of T cells.

Isolation of T cells

T cells are found in both the bloodstream and lymphatic system.Generally, in order to obtain a suitable sample of T cells a venousblood sample is first obtained. In a preferred embodiment of theinvention this blood sample is obtained from the patient requiringtreatment.

The skilled person will be able to prepare a suitable sample of T cellsfor use in the present invention. For example, the sample may be wholeblood, or a sample prepared from blood including, but not limited to,peripheral blood leucocytes (PBLs) or peripheral blood mononuclear cells(PBMC).

The T cells in the blood sample obtained are then be isolated byfluorescent activated cell sorting (FACS). Briefly, this involves theaddition of florescent labels which specifically bind to T cell-specific‘marker’ proteins and sorting the cells into populations based on thepresence or absence of these labels. These fluorescent labels typicallycomprise an antibody, or fragment thereof, to which is attached afluorescent moiety such as phycoerythicin (PE). The choice of label, orlabels, used will determine the cell types present in the sortedpopulations:

Label Used Cell Population isolated Anti CD3 fluorescent label All(cytotoxic and helper) T cells Anti CD8 fluorescent label CD8⁺(cyto-toxic) T cells Anti CD4 fluorescent label CD4⁺ (helper) T cellsAnti CD4 and anti CD25 Regulatory T cells fluorescent labelIn Vitro Transfection T Cells with a Vector Encoding a TCR Specific forthe Target Cell

There are many techniques suitable for the transfection of mammaliancells, such as human T cells, that are known to those skilled in theart. Textbooks including the following provide experimental examplesthat describe the methods involved: Sambrook et al. Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989; Ausubel et al. Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1992; Glover DNA Cloning, I andII, Oxford Press, Oxford, 1985; B. D. Hames & S. J. Higgins Nucleic AcidHybridization 1984; J. H. Miller and M. P. Calos, Gene Transfer VectorsFor Mammalian Cells, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1987).

As stated above two recent patent applications are directed to thetransfection of T cells with TCRs. (WO 01/55366 and US 20020064521) Themethods disclosed in these applications are also applicable to thetransfection of T cells with the TCRs of this invention that comprise adisulfide bond between residues not present in native TCRs. Briefly, WO01/55366 discloses a retro-viral method for the introduction of TCRswith defined specificity into T cells. The application describes methodsfor the production of a retro-viral vector containing the α and β chainsof a high affinity murine TCR specific for a nucleoprotein peptide(ASNENMDAM) (SEQ ID NO:22) presented by the murine class I MHC H-2D^(b).This vector was then replicated in a human embryonic kidney cell lineand the retroviral supernatant was collected to provide the materialrequired for T cell transfection. US 20020064521 describes methods forthe molecular cloning of cDNA of a human p53-specific, HLA restrictedmurine TCR and the transfer of this cDNA to human T cells. α and β chainTCR cDNAs were subcloned separately into a mammalian expression vector.This vector was then transferred into Jurkat cells using standardliposome transfection procedures. Surface expression Of the transfectedTCR was then confirmed by flow cytometry.

In Vitro Growth of the Transfected T Cells

Once the T cells required for adoptive therapy have been transfectedwith the required TCR they can optionally be cultured in vitro toprovide an expanded population of T cells using standard techniques.

One preferred method for the expansion of transfected T cells of theinvention relies on the use of magnetic beads coated with the specificTCR ligand recognised by the introduced TCR, and a combination of antiCD28 and anti-CD3. Briefly, the use of these beads allows the selectiveexpansion of T cells possessing functional transfected TCRs. The beadsare commercially available in an anti-biotin coated form (MiltenyiBiotec, Bisley UK) which can then be coated with the biotinylatedligands of choice. (Example 9 herein details the required methodology)

Once the T cells have been prepared using the above methods they can beadministered to patients together with a pharmaceutically acceptablecarrier.

Administration of the Transfected Cells to the Patient

The invention provides a method of directing cells to a population oftarget cells in a patient, said method comprising administering to apatient a plurality of cells expressing a surface anchored TCR, whereinsaid TCR comprises a disulfide interchain bond between extracellularconstant domain residues which is not present in native TCRs and whereinthe TCR presented by such cells is specific for a TCR ligand on thepopulation of target cells.

The invention also provides a method of directing a T cell response to atarget cell phenotype in a patient, said method comprising administeringto a patient a plurality of T cells expressing a surface anchored TCR,wherein said TCR comprises a disulfide interchain bond betweenextracellular constant domain residues which is not present in nativeTCRs and wherein the TCR presented by such T cells is specific for a TCRligand on the target cell type.

In another embodiment of the invention the TCR ligand on the target celltype is a peptide-MHC complex or a CD1-antigen complex.

In a further embodiment of the invention the administered cells are notcytotoxic T cells.

In a further embodiment of the invention the target cell is a cancercell or infected cell and the administered cells are cytotoxic T cells.

In a further embodiment of the invention the TCR ligand is unique to onetissue-type or to cells characteristic of one organ of the body.

In another embodiment of the invention the target cell is a target forauto-reactive T cells in autoimmune disease, organ rejection or GraftVersus Host Disease (GVHD). In a specific embodiment the target cells isan islet cell.

Examples of suitable MHC-peptide targets for the TCR according to theinvention include, but are not limited to, viral epitopes such as HTLV-1epitopes (e.g. the Tax peptide restricted by HLA-A2; HTLV-1 isassociated with leukaemia), HIV epitopes, EBV epitopes, CMV epitopes;insulin and/or IGRP-derived diabetes epitopes; melanoma epitopes (e.g.MAGE-1 HLA-A1 restricted epitope) and other cancer-specific epitopes(e.g. the renal cell carcinoma associated antigen G250 restricted byHLA-A2). Further disease-associated pMHC targets, suitable for use inthe present invention, are listed in the HLA Factsbook (Barclay (Ed)Academic Press), and many others are being identified.

In a further embodiment of the invention the population of T cells isisolated from a patient to be treated.

T cells expressing the transfected TCRs can be administered to thepatients by a number of routes. For example, i.v. infusion at regularintervals, optionally accompanied by the administration of a cytokinesuch as IL-2.

A further embodiment of the invention provides an infusible orinjectable pharmaceutical composition comprising a plurality of cellsexpressing a surface anchored TCR, said TCR comprises a disulfide bondbetween α and β extracellular constant domain residues which is notpresent in native TCRs together with a pharmaceutically acceptablecarrier.

Such pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection formulations which maycontain suspending agents, anti-oxidants, buffers, bacteriostats andsolutes which render the formulation substantially isotonic with theblood of the intended recipient.

Dosages of the cells of the present invention can vary between widelimits, depending upon the disease or disorder to be treated, the ageand condition of the individual to be treated, etc. and a physician willultimately determine appropriate dosages to be used. For example, aeffective dosage may vary between 10⁵ to 10¹⁰ cells/kg body weight. Thepractice of therapeutic administration by infusion is described in anumber of papers. See, for example (Rosenberg 1988 New Eng. J Med 3191676-1680). The dosage may be repeated as often as appropriate. If sideeffects develop the amount and/or frequency of the dosage can bereduced, in accordance with normal clinical practice.

Additional Aspects

The invention provides a method of treatment of cancer, GVHD, infection,organ rejection, or auto-immune disease comprising administering aplurality of cells presenting at least one αβ T cell receptor (TCR)anchored to the membrane by a transmembrane sequence, said TCRcomprising a disulfide interchain bond between extracellular constantdomain residues which is not present in native TCRs. A specificembodiment is provided wherein the auto-immune disease is a diseaseselected from Rheumatoid Arthritis, Diabetes, Multiple Sclerosis orReactive Arthritis

Another aspect of the invention is provided by the use of a cellpresenting at least one αβ T cell receptor (TCR) anchored to themembrane by a transmembrane sequence, said TCR comprising a disulfideinterchain bond between extracellular constant domain residues which isnot present in native TCRs in the preparation of a medicament fortreatment of cancer, GVHD, infection, organ rejection, or auto-immunedisease.

Cancers which may benefit the methods of the present invention include:leukaemia, head, neck, lung, breast, colon, cervical, liver, pancreatic,ovarian and testicular.

Auto-immune diseases which may benefit the methods of the followinginvention include:

Acute disseminated encephalomyelitis

Adrenal insufficiency

Allergic angiitis and granulomatosis

Amylodosis

Ankylosing spondylitis

Asthma

Autoimmune Addison's disease

Autoimmune alopecia

Autoimmune chronic active hepatitis

Autoimmune haemolytic anaemia

Autoimmune Neutrogena

Autoimmune thrombocytopenic purpura

Behçet's disease

Cerebellar degeneration

Chronic active hepatitis

Chronic inflammatory demyelinating polyradiculoneuropathy Chronicneuropathy with monoclonal gammopathy

Classic polyarteritis nodosa

Congenital adrenal hyperplasia

Cryopathies

Dermatitis herpetiformis

Diabetes

Eaton-Lambert myasthenic syndrome

Encephalomyelitis

Epidermolysis bullosa acquisita

Erythema nodosa

Gluten-sensitive enteropathy

Goodpasture's syndrome

Guillain-Barre syndrome

Hashimoto's thyroiditis

Hyperthyroidism

Idiopathic hemachromatosis

Idiopathic membranous glomerulonephritis

Isolated vasculitis of the central nervous system

Kawasaki's disease

Minimal change renal disease

Miscellaneous vasculitides

Mixed connective tissue disease

Multifocal motor neuropathy with conduction block

Multiple sclerosis

Myasthenia gravis

Opsoclonus-myoclonus syndrome

Pemphigoid

Pemphigus

pernicious anaemia Polymyositis/dermatomyositis

Post-infective arthritides

Primary biliary sclerosis

Psoriasis

Reactive arthritides

Reiter's disease

Retinopathy

Rheumatoid arthritis

Sclerosing cholangitis

Sjögren's syndrome

Stiff-man syndrome

Subacute thyroiditis

Systemic lupus erythematosis

Systemic necrotizing vasculitides

Systemic sclerosis (scleroderma)

Takayasu's arteritis

Temporal arteritis

Thromboangiitis obliterans

Type I and type II autoimmune polyglandular syndrome

Ulcerative colitis

Uveitis

Wegener's granulomatosis

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated to the fullest extent permitted by law.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES

The following example describes the preparation of the DNA sequences ofFIGS. 3 a and 3 b. This example is usable for the preparation of thecoding sequences of any given αβ TCR including a non-native disulfideinterchain bond.

Example 1 Design of Primers and Mutagenesis of A6 Tax TCR α and β Chainsto Introduce the Cysteine Residues Required for the Formation of a NovelInter-Chain Disulfide Bond

For mutating A6 Tax threonine 48 of exon 1 in TRAC*01 to cysteine, thefollowing primers were designed (mutation shown in lower case):

(SEQ ID NO: 23) 5′-C ACA GAC AAA tgT GTG CTA GAC AT (SEQ ID NO: 24)5′-AT GTC TAG CAC Aca TTT GTC TGT G

For mutating A6 Tax serine 57 of exon 1 in TRBC1*01 or TRBC2*01 tocysteine, the following primers were designed (mutation shown in lowercase):

(SEQ ID NO: 25) 5′-C AGT GGG GTC tGC ACA GAC CC (SEQ ID NO: 26)5′-GG GTC TGT GCa GAC CCC ACT G

PCR Mutagenesis:

Expression plasmids containing the genes for the A6 Tax TCR α or β chainwere mutated using the α-chain primers or the β-chain primersrespectively, as follows. 100 ng of plasmid was mixed with 5 μl 10 mMdNTP, 25 μl 10×Pfu-buffer (Stratagene), 10 units Pfu polymerase(Stratagene) and the final volume was adjusted to 240 μl with H₂O. 48 μlof this mix was supplemented with primers diluted to give a finalconcentration of 0.2 μM in 50 μl final reaction volume. After an initialdenaturation step of 30 seconds at 95° C., the reaction mixture wassubjected to 15 rounds of denaturation (95° C., 30 sec.), annealing (55°C., 60 sec.), and elongation (73° C., 8 min.) in a Hybaid PCR expressPCR machine. The product was then digested for 5 hours at 37° C. with 10units of DpnI restriction enzyme (New England Biolabs). 10 μl of thedigested reaction was transformed into competent E. coli XL1-Bluebacteria and grown for 18 hours at 37° C. A single colony was picked andgrown over night in 5 ml TYP+ampicillin (16 g/l Bacto-Tryptone, 16 g/lYeast Extract, 5 g/l NaCl, 2.5 g/l K₂HPO₄, 100 mg/l Ampicillin). PlasmidDNA was purified on a Qiagen mini-prep column according to themanufacturer's instructions and the sequence was verified by automatedsequencing. The respective mutated nucleic acid and amino acid sequencesare shown in FIGS. 3 a and 4 a for the α chain and FIGS. 3 b and 4 b forthe β chain.

The following example describes the extension of the DNA of FIGS. 3 aand 3 b to add sequences coding for the remainder of the constantdomains of the A6 TCR. Again, this example is usable for the extensionof the constant domain encoding sequences of a corresponding solublevariant of any given αβ TCR.

Example 2 Design of A6 Tax TCR α and β Chain Nucleic Acid SequencesRequired to Produce a Cell Surface Membrane Anchored A6 Tax TCRIncluding Cysteine Residues Required for the Formation of a NovelInter-Chain Disulfide Bond

The constructs encoding the α and β chains of a soluble disulfide-linkedA6 Tax TCR molecule prepared as described in example 1 are used, alongwith cDNA from human peripheral blood mononuclear cells (PBMCs), in theproduction of constructs encoding the α and β chains of a membraneanchored A6 Tax TCR including cysteine residues required for theformation of a novel inter-chain disulfide bond. (Refer to FIG. 5 for adiagrammatic representation of the method involved).

TCR α chain DNA corresponding to Fragment 1 (See FIG. 5 a) is amplifiedfrom cDNA encoding wild-type TRAY 12-2 TCR by PCR using the followingprimer pair specific for the TRAY 12-2 TCR signal peptide (Fwd primer)and the TRAY 12-2 TCR variable domain (Rev primer):

5′ Fwd α primer: (SEQ ID NO: 27) 5′- ATG ATG AAA TCC TTG AGA GTTTT -3′5′ Rev α primer: (SEQ ID NO: 28) 5′- GTA AGT GCA GTT GAGAGAGG -3′

TCR β chain DNA corresponding to Fragment 1 (See FIG. 5 a) is amplifiedfrom cDNA encoding wild-type TRBV 6-5 TCR by PCR using the followingprimer pair specific for the TRBV 6-5 TCR signal peptide (Fwd primer)and the TRBV 6-5 TCR variable domain (Rev primer):

5′ Fwd β primer: (SEQ ID NO: 29) 5′- ATG AGC ATC GGC CTC CTG T -3′ 5′Rev β primer: (SEQ ID NO: 30) 5′- TT CAT ATC CTGGGC ACA CTG -3′

The above primers are designed to produce a PCR product thatincorporates an overlap with the DNA encoding the variable region of thesoluble disulfide-linked A6 Tax TCR produced in example 1.

TCR α chain DNA corresponding to Fragment 2 (See FIG. 5 b) is amplifiedfrom cDNA from PBMC using the following primer pair specific for the 3′end of TRAC, this primer pair also introduces a Cys to Ser mutationdisrupting the formation of the native inter-chain disulfide bond:

3′ Fwd α primer: (SEQ ID NO: 31)5′- TC CCC AGC CCA GAA AGT TCC TCT GAT GTC  AAG CTG GTC GAG AAA AG -3′3′ Rev α primer: (SEQ ID NO: 32) 5′- TTA GCT GGA CCA CAG CCG CAG -3′

TCR β chain DNA corresponding to Fragment 2 (See FIG. 5 b) is amplifiedfrom cDNA from PBMC using the following primer pair specific for the 3′end of TRBC2, this primer pair also introduces a Cys to Ser mutationdisrupting the formation of the native inter-chain disulfide bond:

3′ Fwd β primer: (SEQ ID NO: 33)5′- CC GAG GCC TGG GGT AGA GCA GAC TCT GGC TTC ACC TCC GAG TCT TAC C -3′3′ Rev β primer: (SEQ ID NO: 34) 5′- TTA GCC TCT GGA ATC CTT TCT C- 3′

These primers are designed to produce a PCR product that incorporates anoverlap with the DNA encoding the constant region of the solubledisulfide-linked A6 Tax TCR produced in example 1.

Final PCRs are required to assemble the entire genes for the twoTCR-chains. For the alpha chain fragments 1 and 2 are mixed with theplasmid coding for the soluble alpha-chain and the full length codingregion is amplified using the 5′ Fwd a primer and the 3′ Rev a primerwith suitable restriction site sequences added to the primers asflanking sequences to facilitate sub-cloning in the required vector (forexample, the retroviral pLXSN vector, BD Clontech, UK). The fragment issub-cloned into the expression vector and sequenced.

For the beta chain fragments 1 and 2 are mixed with the plasmid codingfor the soluble beta-chain and the full length coding region isamplified using the 5′ Fwd β primer and the 3′ Rev β primer withsuitable restriction site sequences added to the primer as flankingsequences to facilitate sub-cloning into the required vector (forexample, the retroviral pLXSN vector, BD Clontech, UK). The fragment issub-cloned into the expression vector and sequenced.

FIGS. 6 a and 6 b show the nucleic acid and protein sequences of themembrane anchored α and β chain of A6 TCR respectively, mutated so as tointroduce a new cysteine codon and mutate the cysteine residues involvedin forming the native inter-chain disulfide bridge to Ser.

The above PCR reactions are all carried out using the followingmethodology.

For a 100 μl reaction mix:

-   -   1. 18 MO quality H2O to 100    -   2. 50 pmol Forward Primer    -   3. 50 pmol Reverse Primer    -   4. 2 μl 10 mM dNTP (10 mM each of dATP, dTTP, dCTP, dGTP).    -   5. 10 μl 10× Buffer (Pfu buffer for cloning purposes and Taq        buffer for diagnostic PCR).    -   6. 5 units of enzyme (Pfu DNA Polymerase or Taq polymerase        according to the particular application).

PCR program:

-   -   1. A denaturation step where the sample is heated to 94° for 10        minutes.    -   2. A number of cycles (20-40) including        -   a denaturation step 1 minute @94°        -   an annealing step 1 minute @45-60° (use the gradient block            in PCR-1 if you need to establish the optimal annealing            temperature).        -   an elongation step 5-10 minutes @72-73°.    -   3. A final elongation step 10 minutes @72-73° to ensure that all        products are full length    -   4. followed by a soak step at 4°.

The following example describes the preparation of the DNA sequences ofFIGS. 9 a and 9 b. This example is usable for the preparation of thecoding sequences of any given αβ TCR which includes a non-nativedisulfide interchain bond.

Example 3 Production of DNA Encoding a Soluble AH-1.23 TCR Comprising aNon-Native Disulfide Inter-Chain Bond

Synthetic genes encoding the TCR α and TCR β chains of a soluble AH1.23TCR can be manufactured to order. There are a number of companies whichcarry out this service such as GeneArt (Germany).

FIGS. 9 a and 9 b show respectively the DNA sequences of the α and βchains of a soluble AH-1.23 TCR, mutated so as to introduce a novelcysteine codon (indicated by shading).

FIGS. 10 a and 10 b show respectively the AH-1.23 TCR α and β chainextracellular amino acid sequences produced from the DNA sequences ofFIGS. 9 a and 9 b

The DNA sequences shown in FIGS. 9 a and 9 b can then be sub-cloned intothe required vector containing the DNA sequences of FIGS. 3 a and 3 brespectively in such a way as to replace the DNA encoding thecorresponding extracellular portions of the A6 TCR.

The following example describes a means of preparing DNA sequencesencoding full-length TCRs containing a non-native disulfide interchainbond for use in the current invention. Preferably, said DNA sequenceswill comprise restriction enzyme recognition site to facilitate ligationof the sequences into the vector of choice. This example is usable forthe production of any αβ or γδ TCR.

Example 4 Production of Nucleic Acids Encoding Alternative TCR α and βChains of Membrane Anchored TCRs Including Cysteine Residues Requiredfor the Formation of a Novel Inter-Chain Disulfide Bond

To incorporate DNA encoding an alternative TCR into the vector(s) ofchoice synthetic genes encoding the required full-length TCR α and TCR βchains, altered in order to encode the required introduced cysteineresidues in the constant domains thereof, can be manufactured to order.There are a number of companies which carry out this service such asGeneArt (Germany). Such DNA sequences can be produced which incorporaterestriction enzyme recognition sequences to facilitate ligation of theDNA produced into the vector of choice.

For transfection of the desired cells with the expression vectorsprepared according to example 6, selection of the appropriate vector isrequired:

Example 5 Vector Choice for the Transfection of T Cells with DNAEncoding TCRs Containing Cysteine Residues Required for the Formation ofa Novel Inter-Chain Disulfide Bond

As will be obvious to those skilled in the art the primary differencebetween transient and stable transfection methods is the choice ofvector. The following table provides a summary of a number of vectorssuitable for the transient transfection and/or stable transfection of Tcells with DNA encoding TCRs containing cysteine residues required forthe formation of a novel inter-chain disulfide bond:

Random Stable Site- Stable Vector and Transient Stable Specific EpisomalSupplier Expression Integration Integration Maintenance pCI ✓  ✓* x x(Promega) pCI_(neo) ✓  ✓* x x (Promega) pREP4 ✓ x x ✓ (Invitrogen) pCEP4✓ x x ✓ (Invitrogen) pcDNA5/FRT ✓ (✓) ✓ x (Invitrogen) FRT ✓ ✓ ✓ xRetroviral Vectors Standard ✓ ✓ x x Retroviral Vectors ✓* Suitable whenin combination with an appropriate vector (e.g. pCI_(neo) with pCI). (✓)Capable of random integration, but designed for site-specificintegration into specialised recipient cells.

Ligation of the DNA sequences encoding a TCR containing a non-nativedisulfide interchain bond, prepared for example, as described inexamples 1 and 2, or examples 3 or 4, into the desired vector or vectorsis required. These vectors may be one of those listed in example 5. Thisexample is usable for the ligation of the coding sequences of any givenαβ or γδ TCR including a non-native disulfide interchain bond into thevector(s) of choice:

Example 6 Ligation of DNA Sequences Encoding a TCR Containing aNon-Native Disulfide Interchain Bond into the Desired Vector

In order to facilitate the insertion of DNA encoding the TCR chains inthe desired orientation into the vector or vectors of choice thevector(s) and DNA encoding the TCR chains should each have the same pairof different complementary ends. To achieve this the desired recipientvector or vectors, and the DNA sequences encoding the TCR chains aredigested with the same appropriate pair of differing restrictionenzymes. The cut DNA chains and the cut vector or vectors are thenligated using the Rapid DNA Ligation kit (Roche) following themanufacturers instructions.

Example 7 describes a general procedure for the isolation of T cellsub-populations for transformation to produce cells in accordance withthe invention.

Example 7 Isolation of T Cell Sub Populations

PBMCs are isolated from venous blood samples using Leucosep® tubes(Greiner Bio-one, Germany) following the manufacturer's instruction. Theisolated PBMCs are washed and used immediately. Freshly isolated PBMCsare washed twice in 10% autologous human serum/RPMI (Gibco BRL).Finally, the cells are re-suspended in RPMI medium.

T cell sub-populations are isolated from PBMCs by FACS using therelevant combination of antibodies in the table below for the T cellsub-population required and the following procedure:

Label Used Cell Population isolated Anti CD3 fluorescent label All(cytotoxic and helper) T cells Anti CD8 fluorescent label CD8⁺(cyto-toxic) T cells Anti CD4 fluorescent label CD4⁺ (helper) T cellsAnti CD4 and anti CD25 Regulatory T cells fluorescent label

Under sterile conditions, the relevant fluorescently-labeled antibodies(0.01 mg/ml final concentration) are incubated with PBMCs (1×10⁷/ml) for30 mins at 37° C., 5% CO₂. Cells are then washed using medium (37° C.),centrifuged for 10 mins at 250×g and the supernatant discarded. Thepellet is re-suspended and the cells are then bulk-sorted by FACS. Theselected T cells are collected in either medium containing 10%autologous serum (for in-vitro culturing), or in the appropriateinfusion medium, such as Hank's balanced buffer solution (Sigma, UK)with 10% autologous human serum albumin for immediate therapeutic use.

Alternatively, the required T cell sub-population may be isolated usingmagnetic beads coated with the same antibodies and antibody combinationsdescribed above.

Minimacs beads, produced by Miltenyi Biotech, are suitable for use inthe isolation of T sub-populations and the manufacturer providesinstructions for their use. This method “positively” selects andisolates the desired T cell sub-population. It is also possible to“negatively” select the desired T cell sub-population. This is achievedby coating the beads with a range of antibodies that will bind to allbut the required T cell population in PBMCs.

Example 8 describes one method, usable in accordance with the invention,of modifying isolated cells for expression of TCRs containing anon-native disulfide interchain bond.

Example 8 Retro-Viral Transduction of T Cell with TCRs ContainingIntroduced Cysteine Residues Capable of Forming a Non-Native DisulfideInterchain Bond

Primary T cells or T cell lines/clones are transduced with anappropriate retroviral vector, (e.g. the pLXSN retrovirus (BD ClonTech,UK)) following a T cell transduction methodology based on that describedin (Clay (1999) J Immunol 163 507-513 and Bunnel (1995) PNAS USA 927739)

Production of Retroviral Supernatant

Briefly, in order to produce retroviral supernatant, the PG13 retrovirusproducer cell line is transduced with the retroviral vector (pLXSN, BDClontech, UK) produced in example 2 that contains DNA encoding the α andβ chains of a membrane anchored A6 Tax TCR including cysteine residuesrequired for the formation of a novel inter-chain disulfide bond. Hightitre clones are then isolated using standard techniques familiar tothose skilled in the art. (See, for example (Miller (1991) J Virol 652220) A high titre clone is then grown to 80% confluence and thesupernatant is then harvested.

Transduction of T Cells with Retroviral Supernatant

T cells are then re-suspended at 1×10⁶ cells/ml in microtitre wellplates in retroviral supernatant containing 8 μg/ml polybrene and 600IU/ml IL-2. The plates are then centrifuged at 1000×g for 90 mins andincubated overnight at 37° C. in a humidified 5% CO₂ incubator. Thistransduction procedure is repeated after 2 days. The transductionprocedure described in (Clay (1999) J. Immunol. 163 507-513) is thenfollowed, thereby providing transfected T cells ready for in-vitrotesting.

Example 9 describes a general method for enriching and enlarging apopulation of T cells in accordance with the invention. This method isnot TCR specific.

Example 9 In-Vitro Growth of Transfected T Cells

After the transfection of T cells to express modified TCRs as describedin Example 8 these T cells can, if necessary, be grown in-vitro toproduce an enriched and enlarged populations of cells for in-vitroevaluation or therapeutic use using the following method.

Anti-biotin coated Clinimacs beads (Miltenyi Biotec, Germany) are coatedwith biotinylated anti-CD28 and anti-CD3 antibodies. 500,000 T cells and500,000 autologous irradiated (33 Gy) APCs pulsed with the appropriatepeptide (Tax peptide), are added to RPMI 1640 buffer containing 10-50U/ml IL-2 and 10% autologous serum. 5×10⁶/ml anti-CD28 and anti-CD3antibody coated Clinibeads are then added to the cells.

The cells are then incubated under sterile conditions at 37° C., 5% CO₂for 7 days. During this incubation period the buffer is replaced every 3days. The cells can be re-stimulated the following week with the sameratio of beads to T-cells and fresh peptide-pulsed APCs. Once therequired total number of transfected T cells has been reached the Tcells are then re-suspended in the appropriate buffer for in-vitroevaluation or therapeutic use.

Example 10 describes one method of testing for successful cell surfaceexpression of the desired TCRs on the chosen modified cell. This methodis generally applicable, and not restricted to any particular cellsurface TCR.

Example 10 Fluorescence Activated Cell Sorting (FACS)-Based Assay toDemonstrate Specific Binding of Cognate Peptide-MHC Complexes to T CellsTransfected to Express an A6 Tax TCR Incorporating Cysteine ResiduesRequired for the Formation of a Novel Inter-Chain Disulfide Bond

Preparation of T Cell Samples for Staining

The transfected T cells are re-suspended in FACS staining buffer (2%FCS/PBS, at 37° C.) and counted. The cells are aliquoted into FACS tubesand pre-incubated at 37° C. for 5-10 minutes prior to staining.

Staining of T cells with HLA-A2 Tax Tetramers to Assess TCR-pMHC Binding

In order to stain the transfected T cells HLA-A2 Tax monomers areprepared using the methods described in WO 99/60120, and tetramerisedusing Phycoerythrin (PE)-labelled streptadivin via the methods describedin (O'Callaghan (1999) Anal Biochem 266 9-15)

The following fluorescently labelled molecules are also used in the FACSassay as controls:

FITC-labelled isotype controls

PE-labelled “irrelevant” peptide-HLA-A2 tetramers

PE-labelled HLA-A2 Tax tetramer (48 μg) is incubated with 1×10⁶transfected T cells and 5 μg anti-CD8-FITC labelled antibody (or 5 μganti-CD4-FITC labelled antibody) for 20 mins at 37° C. Cells are thenwashed using FACS buffer (37° C.), centrifuged for 10 mins at 250×g andthe supernatant discarded.

After the wash, transfected T cells are re-suspended in 0.5 ml PBS. TheT cell populations present in the samples are then analysed by flowcytometry.

Any T cells that are double-labelled by both the PE-HLA-A2 Tax tetramersand the αCD8-FITC labels (or anti-CD4-FITC labelled antibody) are CD8⁺ Tcells (or CD4+ T cells) expressing the transfected A6 Tax TCR.

The above HLA tetramer FACs staining method can be adapted to assess theexpression level of any exogenous TCR on the surface of T cells by usingtetramers of the cognate peptide-HLA for the desired exogenous TCR.

Staining of Transfected T Cells with Antibodies to Assess Exogenous TCRExpression

As will be obvious to those skilled in the art there are other bindingagent that can be utilised in such FACS methods, or any other suitabledetection methods, for the assessment of exogenous TCR. The followingtable provides a summary of some antibodies suitable for this purpose:

Antibody Specificity Usage Specific TCR variable domain Assessment ofexogenous TCR expression (e.g. anti-Vβ30) on T cells posessing anendogenous TCR of differing V domain usage Pan TCR Assessment ofexogenous TCR expression on TCR- cells CD3 Assessment of exogenous TCRexpression on CD3− TCR- cells. The presence of the exogenous TCR should“rescue” cell surface CD3 presentation

Example 11 describes one method of testing for successful cell surfaceexpression of functional exogenous TCRs on the surface of a CTL. Thismethod is specific for such CTL cells. However, the method is notlimited to a specific TCR.

Example 11 Europium-Release Method for Assessing the Ability of CTL‘Killer’ T Cells Transfected to Express the Membrane-Anchored A6 Tax TCRto Specifically Lyse Target Cells

The following assay is used to assess the ability of CTLs transfected toexpress the membrane-anchored A6 Tax TCR to specifically lyseHLA-A*0201⁺ target cells.

The following mixtures are prepared for the assays:

-   -   Experimental wells: 50 μl of Transfected CTLs, 50 μl of media,        50 μl targets cells pulsed with the cognate HLA-A2 peptide.    -   Negative control wells: 50 μl of Transfected CTLs (effector        cells), 50 μl of media, 50 μl targets cells pulsed with an        irrelevant HLA-A2 peptide.    -   Background wells: APC Target cells are spun down after dilution        to final concentration and the 50 μl of supernatant added to 100        μl media.    -   Spontaneous release wells: Target cells alone (no effector        cells)+100 μl media    -   Maximum release wells: spontaneous release wells +15 μl of 10%        Triton (Sigma T-9284)

Briefly, the above mixtures of effector and target cells are incubatedfor 2 to 4 hours and the Europium release assay is then carried outfollowing the instructions supplied with the Delfia EuTDA CytotoxicityKit (Perkin Elmer).

Example 12 describes one method of testing for successful cell surfaceexpression of functional exogenous TCRs on the surface of regulatory Tcells or CTLs. The method is not limited to a specific TCR.

Example 12 Thymidine Incorporation Assay for Assessing the Ability of TCells Transfected to Express the Membrane-Anchored AH1.23 TCR toSpecifically Alter T Cell Proliferation

5×10⁶ PMBCs are pulsed with 1 μM of the cognate peptide for the AH1.23TCR and then cultured in RPMI 1640 medium at 37° C., 5% CO₂ for 14 days.A control group of 5×10⁶ PMBCs cultured at 37° C., 5% CO₂ for 14 dayswithout peptide pulsing. Both cultures are fed with 40 units/mlrecombinant human IL-2 every 3 days.

The following are then added to 1×10⁵ cells in a 96 well plate both thecultures prepared above:

-   -   1×10⁵ fresh autologous irradiated (33Gy) PBMCs, and a range (0        cells, 5×10⁴, 1×10⁵, 2×10⁵, 5×10⁵) of T cells transfected with        the AH1.23 TCR using the methods described in the previous        examples. These cultures are then incubated in RPMI 1640 medium        for 3 days at 37° C., 5% CO₂.    -   1.85 MBq/ml of H³ Thymidine is then added to these cultures and        they are incubated for a further 8 hours at 37° C., 5% CO₂. The        cells are harvested using a cell-harvester, and the level of        thymidine incorporation into the cells is measured using a        TopCount scintillation counter.

A reduction in thymidine incorporation into the previouslypeptide-pulsed PBMCs, compared to that seen in the non-pulsed PMBCsindicates that the transfected Regulatory T cells are causing apMHC-specific down-regulation of cell proliferation.

An increase in thymidine incorporation into the previouslypeptide-pulsed PBMCs, compared to that seen in the non-pulsed PMBCsindicates that the transfected CTLs are causing a pMHC-specificup-regulation of cell proliferation.

Example 13 describes the treatment of patients with cells in accordancewith the invention. This treatment method can be used for T cellstransfected with any exogenous TCR.

Example 13 Infusion into Patients of T Cells Transfected to Express TCRsIncluding Cysteine Residues Required for the Formation of a NovelInter-Chain Disulfide Bond

In order to infuse the transfected T cells expressing TCRs includingcysteine residues required for the formation of a novel inter-chaindisulfide bond into patients the following methodology, as described in(Hague (2002) Lancet 360 436-442), is used. Briefly, the transfected Tcells are washed in Hank's balanced buffer solution (Sigma, UK) with 10%autologous human serum albumin and then re-suspended in 20 ml of thesame buffer solution. The transfected T cells are then slowly infusedinto the patient requiring treatment at a dose of 10⁶ cells per kgbodyweight over a 15 minute period. The patient's vital signs areregularly checked over the next 4 hours to detect any toxic effects.

These infusions are then repeated periodically, and the condition of thepatient assessed by the most appropriate method. For example, in thecase of a patient receiving the transfected TCRs as a means of treatinga tumour these could include one or more of the following palpation,radiography, CT scanning or biopsy. The dosage and frequency of theinfusions is varied if required. Finally the outcome of the treatment at6 months after the final infusion is also recorded in accordance withWHO criteria.

The invention claimed is:
 1. A method for obtaining modified mammaliancell, wherein the modified mammalian cell comprising a membranepresenting at least one modified T cell receptor (TCR) anchored to themembrane by a transmembrane sequence, the modified TCR comprising aninterchain disulfide bond between extracellular constant domain residueswhich is not present in native TCRs, which method comprises incubatingmammalian cell containing an expression vector encoding the modified TCRunder conditions whereby the modified TCR is expressed, therebyobtaining the modified mammalian cell.
 2. A method of directing immunecells to a population of target cells in a patient, said methodcomprising administering to the patient a plurality of immune cellscomprising a membrane presenting at least one modified TCR anchored tothe membrane by a transmembrane sequence, wherein the modified TCRcomprises an interchain disulfide bond between extracellular constantdomain residues which is not present in native TCRs wherein the modifiedTCR presented by such cells is specific for a TCR ligand presented onthe target cells thereby directing the cells to the population of targetcells in the patient.
 3. A method of directing a T cell response to atarget cell in a patient, said method comprising administering to thepatient a plurality of T cells comprising a membrane presenting at leastone modified TCR anchored to the membrane by a transmembrane sequence,wherein the modified TCR comprises an interchain disulfide bond betweenextracellular constant domain residues which is not present in nativeTCRs, wherein the modified TCR presented by such T cells is specific fora TCR ligand presented on the target cell, thereby directing the T cellresponse to the target cell in the patient.
 4. The method according toclaim 2 wherein the TCR ligand presented on the target cells is apeptide-MHC complex or a CD1-antigen complex.
 5. The method according toclaim 2 wherein the administered cells are not cytotoxic T cells.
 6. Themethod according to claim 2 wherein the target cell is a cancer cell orinfected cell and the administered cells are cytotoxic T cells.
 7. Themethod according to claim 2 wherein the TCR ligand is unique to onetissue type or to cells characteristic of one organ of the body.
 8. Themethod according to claim 7 wherein the target cell is a target forautoreactive T cells in autoimmune disease, organ rejection or graftversus host disease (GVHD).
 9. The method according to claim 8 whereinthe target cell is an islet cell.
 10. A method for the preparation ofcells comprising a membrane presenting a plurality of modified (TCRs)anchored to the membrane by a transmembrane sequence, wherein themodified TCRs comprise an interchain disulfide bond betweenextracellular constant domain residues which is not present in nativeTCRs, said method comprising: (a) isolation of a population of cells (b)in vitro transfection of said population of cells with an expressionvector encoding the modified TCRs, said TCRs being specific for a targetcell, and optionally (c) in vitro growth of the transfected cells. 11.The method according to claim 10 wherein the cells are T cells.
 12. Themethod according to claim 10 wherein the population of cells is isolatedfrom a patient who is to be treated by directing immune cells comprisinga membrane presenting at least one modified TCR anchored to the membraneby a transmembrane sequence, wherein the modified TCR comprises aninterchain disulfide bond between extracellular constant domain residueswhich is not present in native TCRs wherein the modified TCR presentedby such cells is specific for a TCR ligand on the population of targetcells to a population of target cells in the patient.
 13. A method oftreatment of cancer, GVHD, infection, organ rejection, or autoimmunedisease comprising administering a plurality of immune cells comprisinga membrane presenting at least one modified TCR anchored to the membraneby a transmembrane sequence, wherein the modified TCR comprises aninterchain disulfide bond between extracellular constant domain residueswhich is not present in native TCRs.
 14. The method according to claim13 wherein the auto-immune disease is rheumatoid arthritis, diabetes,multiple sclerosis or reactive arthritis.
 15. The method according toclaim 3 wherein the TCR ligand on the target cell type is a peptide-MHCcomplex or a CD1-antigen complex.
 16. The method according to claim 3wherein the administered cells are not cytotoxic T cells.
 17. The methodaccording to claim 3 wherein the target cell is a cancer cell orinfected cell and the administered cells are cytotoxic T cells.
 18. Themethod according to claim 3 wherein the TCR ligand is unique to onetissue type or to cells characteristic of one organ of the body.
 19. Themethod according to claim 18 wherein the target cell is a target forautoreactive T cells in autoimmune disease, organ rejection or (GVHD).20. The method according to claim 19 wherein the target cell is an isletcell.